This application relates to a device and a method for rapid hygiene testing by detecting ATP found in biomass on a test surface, such as a food preparation surface. From a sample taken from the surface, ATP is detected using a luciferase/luciferin bioluminescent reaction. The device and method of the present invention provides a quick, accurate determination of the cleanliness of surfaces.
It is important in many industries, such as food preparation, medicine, beverages, toiletries, and pharmaceuticals, to provide clean and sanitary surfaces. It is not enough to simply clean or sanitize a surface and assume it is free from microorganisms such as bacteria. Instead, a test must be performed to detect whether the surface is actually free of microorganisms. Thus, random areas of a surface, such as a food preparation surface, are tested for microorganisms to determine the general cleanliness of the surface.
One of the oldest methods to check for cleanliness involves culturing samples for bacteria. A test surface is chosen and wiped with a swab, and then the swab is smeared onto a culture medium. The medium is incubated and then checked for the presence of bacteria colonies grown in the medium. Over the years, various types of culture media have been developed along with numerous products based thereon. While the results of bacterial cultures are accurate, they are limited by the time that it takes to incubate the culture, usually in the order of days.
In response for a need to obtain results more quickly, other methods for detecting microorganisms were developed. Research soon focused on the detection of biomass on the test surface. Biomass includes living cells, dead cells, other biotic products such as blood, and food residue. It was discovered that biomass could be detected by detecting ATP, adenosine triphosphate, a chemical found in all living organisms.
The specific test for ATP involves the "firefly" reaction. The following is the reaction: ##EQU1##
ATP, luciferin (D-luciferin cofactor), luciferase (enzyme) and oxygen are reacted in the presence of magnesium ion. Luciferin and luciferase are the same cofactor and enzyme present in fireflies that yields their namesake light. The products of the reaction are AMP (adenosine monophosphate), inorganic phosphate, carbon dioxide, and light (photons). The reaction, just as in fireflies, produces light. This light can be quantified and used to correlate to an amount of ATP. However, the amount of ATP does not necessarily relate directly to the number of microorganisms or bacterial cells or colonies. In fact, ATP may be from non-microbe biomass such as beef blood; thus the amount of ATP would not be related to microorganisms.
The lack of correlation may be due to the variation in ATP concentration within cells and the degradation of ATP in dead cells. ATP is found in all living cells, but the amount of ATP in cells can vary significantly. For example, prokaryotic cells have about one hundredth the amount of ATP as eukaryotic cells and different strains of bacteria will contain significantly different amounts of ATP. In addition, if a cell is growing or about to divide, it will contain more ATP than a dormant cell. Furthermore, cells that have just died contain ATP and even dead cells may contain ATP. In dead cells, any ATP present may degrade, often caused by a reaction between ATP and intracellular enzymes contained within the dead cells. All of these variables in ATP concentration mean that ATP testing is limited as a means to quantify the number of microorganisms or bacterial cells or colonies. However, ATP testing remains a method to qualitatively determine the presence of biomass, including microorganisms or bacteria.
Thus, the detection of ATP can be used to determine the presence of biomass, whether viable or nonviable. The ability to detect nonviable biomass is important, for example, in testing a surface for cleanliness because nonviable biomass (dead cells) such as food residue can provide a medium for living cells to grow.
Typically, the luciferase, luciferin, and magnesium ion are sold as a single combined reagent, not as individual reagents. The luciferase must be at the proper pH of 7.8 in order to be effective, usually achieved by employment of a buffer solution. If the proper pH is not mainined, the reaction will not work efficiently, and the results will be erroneous. However, luciferase is unstable while in solution, and will degrade, particularly at higher temperatures. Generally, at room temperature, the luciferase solution will remain effective for a period of hours whereas at near freezing temperatures, the luciferase solution will last for a period of days. In addition, luciferin in solution is light sensitive. Light causes the dissolved luciferin to degrade. Once the luciferin has degraded, no cofactor remains to unleash the bioluminescent reaction resulting in false negatives.
To prevent degradation, the luciferin and luciferase can be dried and protected from light. Methods for drying include, but are not limited to, freeze drying and lyophilization. The luciferase is more stable if kept out of solution. When ready to use, the dried luciferin and luciferase are dissolved in water containing an appropriate buffer to form an aqueous solution having the proper pH.
By mixing the luciferase/luciferin reagent with a sample taken from a test surface, extracellular ATP is immediately reacted and detected. However, intracellular ATP cannot be detected unless the ATP is first ectracted from within the cells. Typically, this is accomplished by mixing the sample with an extraction reagent (releasing reagent) which extracts the ATP from within the cells. The extracted ATP then can be mixed with the luciferase/luciferin reagent to produce the observable reaction. It is important that the extraction reagent chosen does not inactivate the luciferase/luciferin reagent. Nor should the extraction reagent be toxic if it is used on a food preparation surface, for example.
The luciferase/luciferin reagent cannot be stored with the extraction reagent as it will inactivate the luciferase and/or the luciferin over time. If either is inactivated, no light will be produced when combined with ATP. Therefore, the luciferase/luciferin reagent and extraction reagent must be stored separately until the time the test is conducted.
The bioluminescent reaction of ATP and luciferase/luciferin has traditionally been conducted using two basic types of systems: vial systems and all-in-one swab devices. A vial system uses a series of vials containing the reagents necessary to conduct the ATP tests. An all-in-one swab device provides all of the reagents and the swab in a self-contained apparatus.
In a vial system, for example, a first vial contains the extraction reagent, a second vial contains dried luciferase/luciferin reagent, and a third vial contains a buffered solution. At the time of the test, the luciferase/luciferin reagent is hydrated by adding the appropriately buffered solvent from the third vial to the vial containing the luciferase/luciferin reagent.
A sample is collected by wiping a pre-wetted swab across the testing surface. Typically, the swab is pre-wetted with saline. The swab containing the sample is placed in a test tube. Next, the proper amount of extraction reagent from the first vial is pipetted into the test tube containing the swab. After sufficient time has passed to ensure ATP extraction, the buffered solution containing hydrated luciferase/luciferin reagent is pipetted into the test tube and the luciferase is allowed to react with the ATP. The test tube is then placed into a luminometer where the amount of light produced by the reaction is measured. If more than one sample is taken, each sample is placed in its own test tube.
While vial systems produce correct results, there are deficiencies. One large problem is that the quantity of luciferase/luciferin solution prepared must be used within a short time period. If leftover solution is saved for later tests, the luciferase will degrade and ultimately become ineffective thus producing no reaction even in the presence of ATP. This problem is compounded by commercial producers of the luciferase/luciferin reagent that only sell the reagent in quantities that produce an amount of solution that is greater than that needed for individual tests. Furthermore, the dried luciferase/luciferin reagent is relatively costly. Thus the vial system results in waste of expensive reagents when only an individual test is required.
Another shortcoming of vial systems is that accurate pipetting and mixing of reagents is required. A pipette is used to transfer the reagents from vial to vial or vial to tube. While pipetting is accurate, it is laborious and time consuming. Further, if any of the vials or pipettes are not sterile, the biomass contained in them will produce a false positive for the presence of ATP.
The all-in-one swab devices apply the same reaction as the vial systems but keep all of the reagents and swab in a self-contained apparatus that fits into a luminometer. More specifically, the all-in-one devices typically involve a swab that is placed in a plastic tube containing several chambers. An advantage to this system is that a unit dose of each reagent is provided for one test, thus avoiding waste of reagents when only one test is required. However, a certain procedure must be followed using an all-in-one device to ensure that the reagents are combined at the appropriate times.
In a typical all-in-one device, a swab pre-wetted with a wetting solution is placed in a sealed tube until ready for use. The wetting solution may contain an extractant. The sealed tube prevents evaporation of the wetting solution. At the appropriate time, the device is opened, the pre-wetted swab is removed, and a sample is collected by wiping the swab along the testing surface. If present, the extractant will extract intracellular ATP from the sample collected on the swab. The swab is then placed back in the tube and the tube is resealed and ready for ATP reaction with the luciferase/luciferin reagents.
Dried luciferase/luciferin reagents are kept in a dry, stable state in the tube until mixed with a buffer solution. The luciferase/luciferin may be kept isolated from the wet swab by placing the luciferase/luciferin in a separate chamber in the tube which can be broken to expose the luciferase/luciferin to the buffer solution. Alternatively, the luciferase/luciferin may be in the form of a pellet that can be placed in a sealed container or can be stuck to the bottom of the tube.
A sealed chamber at one end of the tube contains the buffer solution. The tube is squeezed to break the barrier wall between the chamber and portion of the tube containing the swab, resulting in release of the buffer solution. The tip of the tube is then shaken to allow the luciferase/luciferin reagents to mix with the buffer solution, hydrate, and mix with the sample on the swab. The entire tube is then placed in a luminometer where the amount of light produced is measured.
While the all-in-one systems have overcome many of the problems of the vial systems, they have other shortcomings. For example, all-in-one systems are costly to manufacture since a complex tube arrangement is needed that is resealable and contains a breakable chamber to hold the buffer solution and possibly a second breakable chamber to hold the luciferase/luciferin.
Whatever system is used, the swabbing of the test surface should not itself contaminate the test surface. Thus, for example, the extracting agent used on the swab should not contain toxic chemicals that will leave toxic residues on the test surface.
Again, whatever system is used, the resulting tube containing the luciferase/luciferin and ATP is placed in a lumninometer to read the light produced during the reaction. In the past, luminometers were designed with detectors aimed perpendicular to the axis of the sample tube so that when the sample is inserted in the luminometer's sample measurement chamber, the detector views the light produced by one side of the sample. Side-viewing luminometers are not a problem if ATP in solution is measured. Side-viewing detectors can be a problem if the sample being measured is absorbed onto a swab so that the light emitted is located on only one side of a swab, and that side is placed on the opposite side of the detector, then the amount of light reaching the detector will be less. Thus, the quantitative light measurement becomes dependent upon how the sample is placed in the luminometer.
More recently, a luminometer with a bottom-reading detector was developed which avoids the problems of side-viewing luminometers. A bottom-reading luminometer views the bottom of the sample tube and provides an accurate reading independent of the orientation of the sample, and whether the sample is in solution or absorbed onto a swab.
It is the object of the present invention to provide an accurate test for ATP with the convenience of the all-in-one system but with reduced unit cost. A further object is to provide an all-in-one system requiring a minimal amount of mixing and preparing of reagents. A further object of the present invention is to avoid waste of expensive reagents. A further object is to provide an accurate test that does not contaminate the tested surface. Finally, the invention will provide a system that can be used with either a side-viewing or a bottom-viewing detector.